The apparent low infectivity of HIV-1 is purportedly explained by virion-cell interactions that take place prior to viral entry in which the virus must first diffuse to the cell surface and bind to specialized receptors; these interactions are speculated, from in vitro studies, to be the rate-limiting step involved in the infection process yt the underlying mechanisms are not completely understood. Moreover, receptor binding, in addition to nonspecific interactions limiting infectivity, may dictate the subsequent steps, i.e., entry pathways, of infection. The long-term goal is to discern the mechanisms, at the molecular level, that underlie this infectivity and if they can be employed for novel therapeutic strategies o intervene viral entry and pathogenesis. The overall objective of this application, which is the next principal step for achieving the long-term goal, is to determine how HIV-1 locates and eventually binds to surface receptors as a precursor to viral entry. The central hypothesis is that HIV-1's infectivity, with respect to CD4+ T cells, is due to the combination of low and heterogenous cell receptor densities together with inadequate spike proteins per virion; this will necessitate a diffusional search near the cell surface to locate receptor-rich regions. The rationale for the proposed research is to explain HIV-1's receptor-finding process so as to understand and predict how certain drugs will impact viral entry, possibly inhibiting infection altogether. In order to test the central hypothesis and, thus, accomplish this application's objective, the following three specific aims will be pursued: (i) Characterize the heterogeneity of virions by the number of functional envelope proteins. The number of functional envelope proteins incorporated into individual virions is posited to be correlated with HIV-1 infectivity. Spike proteins on optically trapped virions labeled with fluorescent monoclonal antibodies will be measured using two-photon excitation microscopy. (ii) Elucidate the receptor-finding process with 4D imaging. It is postulated that increasing cell receptor densities and/or envelope expression accelerates the search process and promotes specific attachment of the virion. Real-time single virus tracking will be employed to quantify the interactions between HIV-1 viruses incorporating free mCherry fluorophores and multiple cell lines. (iii) Determine how the internalization process is influenced by viral binding to receptors. Deficient receptor densities and nonspecific interactions may have a quantifiable effect on virion immobilization at the cell surface which results in low internalization efficiency. Following virion attachment, time-lapse imaging will measure the internalization efficiency and differentiate the entry mechanism utilized to ascertain how they are correlated with the receptor-finding process. The contribution here will be a detailed understanding of the receptor-finding process and how it is correlated with HIV-1 internalization. This contribution is significant because investigation of virion-cell interactionswill elucidate the biological mechanisms that underlie HIV's low infectivity and, as a result, offer greater insight in developing therapeutic approaches to intervene viral entry and pathogenesis.